Method of promoting the propagation and the activity of microorganisms



April 9, 1940. Y' 7 2,196,361

METHOD OF PROMOTING THE PROPAGATION Ayn THE ACTIVITY OF MICROORGANISMSFiled July 23, 1937 Fig.1. 2

1 Fig.2

2 '3 i E i 1 ig 3 4 3 I T Fig.3

Patented Apr. 9, 1940 UNITED STATES 2496361 v METHOD OF PROMOTING THErnoPAGAA 'IION ORGANISMS AND THE ac'nvrrr F mono:

Paul Liebesny and Hugo Wertheim, Vienna,

Austria 1 I Application July 23, 1937, Serial No. 155,312 I In AustriaJuly 31, 1938 14 Claims.

This invention, which involves a modification and further development ofthe method described in the co-pending applications Serial Nos. 663,642filed March 30, 1933, now Patent 2,107,830 issued Feb. 8, 1938, and30,934, filed July 11, 1935, now Patent No. 2,133,103, issued Oct. 11',1938, relates to promoting the propagation and activity ofmicroorganisms, and is a continuation-in-part of said copendingapplications.

It is known to iavorably influence microorganisms by irradiation withelectromagnetic waves of a wave length between 120 meters and 1.8millimeters.

For the purposes of the present specification the term short waves is tobe understood as includingwaves of wave-lengths between 120 meters and 6meters, the term ultra short waves" as including wave lengths between 6meters and 2 meters and the term extremely short waves as includingwaves between 2 meters and 1.8 milli= meters. It is also known toinfluence microorganisms by irradiating the organisms (startingorganisms) and causing the same to develop in an inoculation or in aseries of inoculations obtained by successive inoculation from one batchto the other; and also irradiating one or some or all of theinoculations. The terms "inoculation and inoculations are herein used tomean the stages of development or propagation separated by operations ofinoculation from one batch of substrate to another, or the batches ofmash used as substrate. According to the methods hitherto known thequantity of material to be irradiated at any one time, whether suchquantity was large or small, was irradiated as such and in its entirety,the procedure being to introduce the vessel containing the entirequantity of material to be irradiated, into the condenser field of ashort wave emitter. In view of the fact that on account of theseconditions it was only possible to.

irradiate more or less small batches these prior processes have notproved to be satisfactorily applicable to large scale working. Themethod according to the present invention has the great advantage ofenabling any quantity of material to be irradiated at a time, andtherefore of rendering short wave irradiation unrestrictedly applicableon an industrial scale.

The new method according to the present invention consists in carryingout the irradiation of the starting organisms and/or of theinoculations, or of the starting organisms and/or a selection of theinoculations, while the material under treatment is flowing. 4

As in the case of the methods according to the above-mentionedco-pending applications, there are used in the present methodelectromagnetic waves of a wave length between 120 meters and 1.8millimeters. It has proved to be particularly suitable, when working inaccordance with the present invention, to employ wave lengths betweenmeters and 56 centimeters, Incertaln instances it may well proveadequate to irradiate the starting organismsfor a single inoculationselected from a sequence of. inoculations, and to 5 carry out suchirradiation while the material (starting organisms, inoculation batch)is in a state of flux. In certain cases it isadvisable, when workingaccording to the present method, to irradiate not fermentingmashes butmicrol6 organisms suspended in water or the like, while flowing. Theterm inoculation as used in the later part of this specification istherefore to be understood as meaning" not only the iermenting mash atany one stage in'fthe processof propaga- .15 tion by successiveinoculations from one batch to the next but also anysuspension ofmicroorganisms prepared for the inoculation of a batch of mash or othersubstrate. Thus, for instance,

in the application oiithe present methodin the yeast industry, it ispossible to irradiate in suitable containers the culture obtainedbyinocula tion from the starting pure culture, andfiin addition such ofthe subsequent inoculations to be developed for propagation purposesgasconstitute 5 smaller quantities, while such of the inoculations asconstitute larger batches are irradiated under conditions of flow. Q

The present method enables fermenting, prOcv esses to be promotedinevery respect. For. in stance, the growth of the,ferinentationexciting organism and the course. of v fermentation are ca-, pable ofbeing promoted,- and 'thegfyield of product increased, by.,,'the"present ff method. Since, as already pointed out, 'both ferinentingmashes and also microorganisms inj'suspension may be irradiated whileflowi risgthe newfmethod is readily applicablejinf every branch. of thefermentation industry. Eor }instance, in 1 the production of acetoneandbu yl-alcohol by fermen 40 tation, or in lacticacid"fermentation}. einjthe manufacturing of lactic f' (acid, the fermenting 'mashes may beirradiated whilegflowing, and in the manufacturing joffc'oinpressed,yeast ,the mashes or alternatively thesuspended yeast may be irradiatedwhile flowing. 7

Some series ofexperimentsqled to, the. unexpected result that in thecaseoi yeast particularlyfavorable results are attainable when; theirradiaq tion is carried out in accordance-withthe Haven? tion, h is toayyuhen..thes ar i a gani ms.

(starting yeast) and a numbengof;consecutively; developedinoculationsare irradiate t at rest or while flowing and the orinoculations {,inf lth'e series batches irradiated} while flowing ofexerting a favorablednflu which when applied to the compressed yeast fbable improvemen uct (durability, raising power), is preferably carriedout in such a manner that a violent irritating action is first exertedupon the yeast cell by irradiation (preliminary irradiation), and thensubjecting the thus pre-treated (sensitized) yeast to subsequentirradiation in a stage of propagation in which it is necessary toirradiate under flux. The step of exerting irritation has proved to bean extremely effective pre-treatment. It is of advantage to make theirritation to be exerted upon the yeast cell so powerful that the yeastis checked in its growth; the injury must not of course be carried tosuch a point that the viability is destroyed and the yeast is incapableof recovering. On the contrary, the conditions of working must be soadjusted that the growth of the yeast is merely checked for a time.During the preliminary irradiation the material may either be at rest orin a state of flux.

The desired sensitizing may be attained or assisted by the applicationof relatively high voltages. It is not possible to give definitenumerical indications of the voltage to be employed, however, since theabsolute voltage in the fleld can not be measured with the means atpresent available for this purpose. How the necessary high voltages areto be obtained is described at a later stage of the specification.

We have found that the employment of shorter wave lengths in conjunctionwith high voltages leads to an increase in the sensitizing effect. Goodresults are obtained with waves below a wave length of about 12 meters.With waves as short as this satisfactory sensitizing results are alsoobtainable without increasing the voltage to a particularly greatextent.

As a general rule it may be stated that the sensitizing should beefiected to such a pitch that the desired biopositive effect is achievedby the subsequent irradiation.

The preliminary irradiation may be carried out in such a manner that thestarting yeast or two or more inoculations from a sequence of inoculatons, are irradiated. Alternatively, Yeast, into which fresh mash isallowed to flow at intervals, is irradiated once or several times.

The subsequent irradiation of yeast is eifected with one inoculatibn orwith a plurality of inoculations, with the material in flux. Inconnection with this subsequent irradiating, which is carried out underconditions which ensure the attainment of favorable influencing, it isadvisable to aim at intensifying the action by the application of highvoltages. The workis done with such high voltages that the duration ofirradiation can be reduced to seconds. We have found that the subsequentirradiation (after-irradiation) may with. advantage be carried out withwaves of a wave length in excess of about 12 meters.

In order to inhibit the increase in temperature that might otherwiseoccur during irradiation, more particularly during the preliminaryirradiation which is of longer duration, the material is artificiallycooled during irradiation, and that to such an extent that thetemperature is kept under all circumstances under the harmful limit. Wehave found it advisable-to keep yeast, for instance with the -aid ofsuitable temperatured water, at incubation temperature or slightly belowthis temperature.

The suitable conditions of irradiation in any particular instance mustbe ascertained by the experimental selection of the number ofconsecutive inoculation or stages of propagation,

the number and duration of individual irradiations, the distribution ofthe irradiations in respect of the individual inoculations, thethickness of layer of the material irradiated, the charging conditionsin the electrolyte, and so forth. To determine the voltage in thesecondary circuit the spacing of the electrodes from the material to beirradiated is ascertained empirically. The ascertainment of this factor,as of the suitable wave length and other factors, is eflfected for theparticular apparatus used in any one instance. We have found that thevoltage may be increased by various measures, for instance by increasingthe voltage in the primary circuit or by placing the electrodes as farapart as possible. Increasing the spacing of the electrodes isrestricted, however, by the fact that the current intensity also varieswith the spacing of the electrodes.

We have observed that the desired effect can be increased by making theone electrode as small as possible, i. e. so small that the diameter ofthe electrode is considerably smaller than the cross-section of theobject irradiated. In order that the object may be exposed to theirradiation uniformly in its entire extent the other electrode is madeat least as great as the crosssection of the object. The irradiatingaction may be increased also by positioning the material asymmetrically,e. g. as close as possible to one of the electrodes, the other electrodebeing preferably positioned as far away from the flrst as ispracticable.

As a rule, in whatever branch of the fermentation industry theirradiation is practised, the irradiation of flowing material inaccordance with the present invention is carried out on a short sectionof conduit. The conduit itself may be suitably shaped to form a bulb atthe point at which the irradiation is applied, or a special length ofconduit or vessel may be inserted in the conduit at such point. It isadvantageous to shape the irradiation bulb in such a manner that it isof uniform cross-section at right angles to the direction of flow of thematerial to be irradiated while its sides facing the electrodes of theshort wave emitter are disposed parallel to each other. If this were notdone the fermentation exciting organisms flowing through between theelectrodes would not be exposed to the irradiation in a uniformthickness of layer, and the treatment would be uneven in its eifect onthe whole of the material treated. If the bulb has to be differentlyshaped for any reason there may be disposed around it a vat or well ofprismatic or other shape conforming to the above requirements in thematter of shape and crosssection. This well or vat having at least twoparallel walls to be connected to the electrodes of the short waveemitter is filled with a liquid having as nearly as possible the samedielectric constant as the material to be irradiated.

It is advisable to irradiate flowing material as it is flowing from onevessel or tank to another. The duration of irradiation may be varied byaltering the speed of flow and/or by the employment of bulbs ofdifferent cross-sections.

In the accompanying drawing, illustrative of the practising of thepresent invention, Figs. 1 and 2 show a form of construction ofapparatus according to our invention, by way of example. A through flowbulb is shown in Fig. 1 in side elevation and in Fig. 2 in plan view.Fig. 3 shows, diagrammatically how the irradiation bulb is disposed inan existing fermentation plant tomprijsiiig'twd preliminary fermentingvats and ama aI rm tln v t-i The bulb consists ofga prismatic portion Iand twotaperederid portions 2. Parallel to the wider 5 surfaces of'thepart I there are disposed the electrodes To theends' 2 there areconnected the condu'itpipes '14. The'bulb is preferably so positionedtha'tthe' flowingliquid travels therein from below'upwards', andfthatfor the reason that the liquid shouldlfiowthrough the field ofirradiation ass noothly'as possible and with the least possibleeddyjformation. It is also advisable to make thef'bulb of longitudinalshape, and only very g'radujally' to expand the part through which theliquid'isconducted into the condenser field, provided this p'a'rtfhas tobe expanded at all. The shapeof the upper end of the bulb shown in thedrawing' is'necessitated by the fact that it has tobe connected at thisend to a tube of the samedi nensions:as'j'the tube connected to thelowerend of the bulb.

The position of the irradiation bulb in the plant as afwhole depends onthe working conditions and method of working in any particular instance..In thecase of compressed yeast production the bulb may be interposed ina pipe line leadingfrom aye'ast'dissolving vat to a fermentin'g'v-atorin'a separate pipe line through which the-fermenting mash flows. Inthe case of the so production of'acetone and butanol the bulb isinterposed, as shown by way of example in Fig. 3, in thepipe; line 5'leading from the preliminary fermenting'vats sftd the main fermentingvat I. Withithe arrangement shown in the drawing thetotalquantity'ofmaterlal subjected to preliminary.fermentation passesthrough the bulb.

Thedescribed bulbs are made from material which is""pervious,- as nearlyas possible without loss,' to" "the"electroinagnetic waves used. Ex 40jamplesjof"materialsf of this description are glass ceramic "materials;or special compositions or materials 'produced'for' these purposes.

.While we havehereinabove set forth our inventionfin' general terms andso broadly that thosefs'killediinthefart will know how to apply it, we'als'o append the following examples in which ourv invention is appliedto various organisms various'media and in various wave I H ur'iiivention but simply as illustr'ations bfce'rtaii i ways of applyingit.

" of the invention is described in the Examples *(1-')-Mola'ssesof 8Balling containing appropriate nutrientadditions is inoculated with pure:1 cultureyeast a test-tube. The test tube is v the condenser field of ashort wav emitter 1 .5 kw. output. The irradiation w ith"a 4 meter wavethree times in m'inuteseach time, with such adeating voltage, the anodevoltvoltage, that the maximum ding to the 4 meter wave is I irradiationthe test-tube is sjrnatic cooling bath of approxiscapacity. One of theelectrodes, which is mm. in diameter, is disposed at a approximately /3cm. from the cool- I, "'tiie other-electrode, which is 40 min. -inizliaeterfis spaced 60 cm. from the firstmentioned electrode. 75 that atemperature of 28 C. is maintained inside The cooling is so efiected"the test-tube. After irradiation, the contents of the test-tube aretransferred to a fresh nu trient substrate of the same nature as thatfirst used, in a flask of 300 cc. capacity, after which this fiask isirradiated under the same conditions 5 as the test-tube. The contents ofthe flask were then used to inoculate nutrient substrate in a glassvessel of 5 liters capacity, and irradiation once more carried out asbefore. For the purpose of this latter irradiation the cooling bath issuit- 10 ably increased in size to accommodate the 5 liter vessel, andthe electrode near the cooling vat increased in size to a diameter of210 mm.; otherwise the conditions under which the glass vessel isirradiated are the same as those under which 15 the test-tube wasirradiated, and the flask. The yeast suspension irradiated in the'glassvessel is introduced, after 24 hours, into a pure culture apparatus ofthe type commonly used in yeast factories, and propagated, likewise inthe usual 20 manner, up to the third generation, without repetition ofirradiation. The starting yeast for the fourth generation is irradiated,with the aid of the same emitter, with the 15 meter wave at theappropriate intensity of 280 milliamperes anode 26 current strengthfound for this emitter, in a through-flow bulb. The electrodes used are25 cm. in diameter and spaced 36 cm. apart. The duration of irradiationis such that the particles of the material treated pass through thecona0 denser field in 30 seconds. The starting yeast for the fifthgeneration is irradiated at an intensity of only 200 milliamperes anodecurrent strength, for a shorter time (the particles of the materialtreated traversing the field in 9 sec- 5 onds), but otherwise under thesame conditions as the starting yeast for the fourth generation.Measurement of yield shows an increase of 10% in the fifth generation,as compared with yeast produced in precisely the same way but without 40irradiation. The raising power of the irradiated yeast shows animprovement of 5 minutes.

(2) Same procedure as in Example 1 except for the fact that the durationof fermentation in the fifth generation is shortened by 3 hours as 5compared with the normal duration of fermentation. In spite of thisshortening of the duration of fermentation it is found that the yield isthe same as with the normal duration of fermentation but withoutirradiation. Moreover, the rais- 50 ing power is found to be shortenedby 15 minutes.

(3) Clostridium butyricurn is irradiated in a vessel in the condenserfield of a short wave emitter. The irradiated organisms are inoculatedtwice on to further batches, and these batches 55 irradiated under thesame conditions as the starting organisms, that is to say again in avessel. The latter of the two inoculations is then used to start thepreliminary fermentation; The mash of the'preliminary fermentation (the0 third inoculation) is irradiated in a through flow bulb interposed inthe pipe line leading from the preliminary fermenting vat to the mainfermenting vat. Yield determination after the main fermentation shows anincrease of about 4% in the 65 yield of products of fermentation ascompared with the yield of the same procedure without irradiation.

We claim:

1. The method of promoting the propagation .70 and activity ofmicroorganisms, which consists in irradiating the microorganisms to beinfluenced in a condenser held of. from -1.8 mm. to in. wave length,while. the material irradiated ,is'

flowing, maintaining the temperature below the 76 harmful limit andcausing the thus irradiated microorganisms to develop in a series ofinoculations obtained by consecutive inoculation on to fresh substrates.

2. The method as claimed in claim 1, wherein at least one inoculationselected from the said series is irradiated likewise in a condenserwhile the material of the selected inoculation is at rest, andmaintained at a temperature in the neighborhood of the incubationtemperature of the microorganism.

3. Method of promoting the propagation and activity of microorganisms,which consists in causing the organisms to develop in a series ofinoculations obtained by consecutive inoculation on to fresh substrates,irradiating the starting organisms and at least one inoculation selectedfrom this series in a condenser fieldof a wave length of from 1.8 mm. to120 111. while these irradiated organisms are at rest, and thenirradiating at least one inoculation selected also from the said serieslikewise in a condenser field ranging from 1.8 mm. to 120 m. .wavelength, while the selected inoculation is flowing during eachirradiation period, the temperature being kept in the neighborhood'ofthe incubation temperature of the organism being irradiated.

4. Method of promoting the propagation and activity of microorganisms,which consists in developing the microorganisms to be influenced onnutritive foundations in a series of three to nine consecutiveinoculations and in irradiating in the condenser field of a short waveemitter at least one of these inoculations while the material irradiatedis at rest, and at least one of the inoculations while the materialirradiated is fiowing during each period of irradiation the temperaturebeing maintained in the neighborhood of the incubation temperature ofthe organism being irradiated.

5. Method of promoting the propagation and activity of microorganisms,which'consists in developing the microorganisms to be influenced onnutritive foundations in a series of three to nine consecutiveinoculations, and in irradiating in the condenser field of a short waveemitter at least one of. these inoculations. in several periods whilethe material irradiated is at rest, and at least one of the inoculationswhile the material irradiated is flowing during each period ofirradiation the temperaturebeing maintained in the neighborhood of theincubation temperature of the organism being irradiated.

6. Method as claimed in claim 4 wherein the irradiation of the flowingmaterial is performed while the material is flowing in an upwarddirection.

7. Method of promoting the growth and actiivty of yeast, which consistsin irradiating yeast on a nutritive foundation in a condenser field of awave length ranging from 1.8 mm. to 120 m. at rest and maintained at atemperature in the neighborhood of the incubation temperature of theyeast, causing the irradiated material to develop in a series of threeto nine inoculations obtained by consecutive inoculation on to freshsubstrates, and irradiating at least one of the said inoculationslikewise in a condenser field ranging from 1.8 mm. to 120, in. wavelength while flowing.

8. Method of promoting the growth and activity of yeast, which consistsin developing yeast in a series of inoculations obtained by consecutiveinoculation on to fresh substrates, and in aroaaer irradiating in thecondenser field of a short wave emitter at least one of theseinoculations while the material irradiated is at rest, and at least oneof said inoculations while the material irradiated is flowing, and whilecooling every batch of irradiated material to a temperature of 28 C.

9. Method of manufacturing yeast which consists in developing yeast in aseries of inoculations exerting a powerful checking irradiation upon theyeast cell by preliminary irradiation of at least one inoculation of thesaid series in the condenser field of a short wave emitter with theemployment of high voltage while the material is at rest and maintainedat a temperature in the neighborhood of the incubation temperature ofthe yeast and then exerting a promoting afterirradiation by irradiatingat least one inoculation selected also from the said series likewise inthe condenser field of a short wave emitter while this selectedinoculation is flowing during each period of irradiation maintaining thetemperature of the yeast in the neighborhood of its incubationtemperature.

10. Method as claimed in claim 9, in which the said after-irradiation iseflected at high voltage. v 11. Method of promoting the growth andactivity of yeast, which consists in developing yeast in a series ofinoculations exerting a powerful checking irradiation upon the yeastcell by preliminary irradiation of at least one inoculation of the saidseries in a condenser field ran from 1.8 mm. to 12 m. wave length withthe employment of high voltage while the material is at rest andmaintained at a temperature in the neighborhood of the incubationtemperature of the yeast and then exerting a promoting after-irradiationby-irradiating at least one inoculation selected also from the saidseries in a-condenser field ranging from 12 to 120 meters wave lengthwhile the selected inoculation is fiowing during each period ofirradiation maintaining the temperature of the yeast in the neighborhoodof its incubation temperature.

12. Method as claimed in claim 9 wherein the object irradiated isasymmetrically positioned with respect 'tothe electrodes of said shortwave emitter.

13. Method as claimed in claim '9 in which said short waves radiatingfrom a fine point.

14. The method of increasing the yeast yield in the manufacturing ofcompressed yeast, which consists in irradiating yeast on a nutritivefoundation in a condenser field of a wave length rang ing from 1.8 mm.to 12 meters, with the employment of high voltage, while the materialirradiated is at rest and maintained at a temperature in theneighborhood of the incubation temperature of the yeast, causing theirradiated material to develop in aseries of eight inoculations obtainedby consecutive inoculation on to fresh substrates, and irradiating thefirst two of the said eight inoculations in the same condenser field andlikewise at rest, and irradiating the starting yeast for the fourth andfifth generations of the run in a condenser field of a wave lengthranging from 12 to 120 meters, at high voltage, while the materialirradiated is flowing during each period of irradiation maintaining thetemperature of the yeast in the neighborhood of its incubationtemperature.

PAUL LIEBESNY. HUGO WERTHEIH.

